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      Rickettsial infection in Amblyomma cajennense ticks and capybaras ( Hydrochoerus hydrochaeris) in a Brazilian spotted fever-endemic area

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          Abstract

          Background

          Brazilian spotted fever (BSF), caused by the bacterium Rickettsia rickettsii, is the deadliest spotted fever of the world. In most of the BSF-endemic areas, capybaras ( Hydrochoerus hydrochaeris) are the principal host for the tick Amblyomma cajennense, which is the main vector of BSF.

          Methods

          In 2012, a BSF case was confirmed in a child that was bitten by ticks in a residential park area inhabited by A. cajennense-infested capybaras in Itú municipality, southeastern Brazil. Host questing A. cajennense adult ticks were collected in the residential park and brought alive to the laboratory, where they were macerated and intraperitoneally inoculated into guinea pigs. A tick-inoculated guinea pig that presented high fever was euthanized and its internal organs were macerated and inoculated into additional guinea pigs (guinea pig passage). Tissue samples from guinea pig passages were also used to inoculate Vero cells through the shell vial technique. Infected cells were used for molecular characterization of the rickettsial isolate through PCR and DNA sequencing of fragments of three rickettsial genes ( gltA, ompA, and ompB). Blood serum samples were collected from 172 capybaras that inhabited the residential park. Sera were tested through the immunofluorescence assay using R. rickettsii antigen.

          Results

          A tick-inoculated guinea pig presented high fever accompanied by scrotal reactions (edema and marked redness). These signs were reproduced by consecutive guinea pig passages. Rickettsia was successfully isolated in Vero cells that were inoculated with brain homogenate derived from a 3 rd passage-febrile guinea pig. Molecular characterization of this rickettsial isolate (designated as strain ITU) yielded DNA sequences that were all 100% identical to corresponding sequences of R. rickettsii in Genbank. A total of 83 (48.3%) out of 172 capybaras were seroreactive to R. rickettsii, with endpoint titers ranging from 64 to 8192.

          Conclusions

          A viable isolate of R. rickettsii was obtained from the tick A. cajennense, comprising the first viable R. rickettsi isolate from this tick species during the last 60 years. Nearly half of the capybara population of the residential park was seroreactive to R. rickettsii, corroborating the findings that the local A. cajennense population was infected by R. rickettsii.

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          Most cited references37

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          Phylogenetic analysis of members of the genus Rickettsia using the gene encoding the outer-membrane protein rOmpB (ompB).

          To confirm the phylogenetic analysis previously inferred by comparison of the citrate synthase and rOmpA gene sequences (gitA and ompA, respectively), the rOmpB gene (ompB) of 24 strains of the genus Rickettsia was amplified and sequenced. rOmpB is an outer-membrane protein of high molecular mass, the presence of which can be demonstrated in most rickettsiae by immunological cross-reactivity in Western blots. No PCR amplification was obtained with Rickettsia bellii or Rickettsia canadensis. For the other rickettsiae, phylogenetic analysis was inferred from the comparison of both the gene and derived protein sequences by using parsimony, maximum-likelihood and neighbour-joining methods which gave the same organization. All nodes were well supported (>86% bootstrap values), except in the cluster including Rickettsia africae strain S and Rickettsia parkeri, and this analysis confirmed the previously established phylogeny obtained from combining results from gltA and ompA. Based on phylogenetic data, the current classification of the genus Rickettsia is inappropriate, specifically its division into two groups, typhus and spotted fever. Integration of phenotypic, genotypic and phylogenetic data will contribute to the definition of a polyphasic taxonomy as has been done for other bacterial genera.
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            Ecology of rickettsia in South America.

            Until the year 2000, only three Rickettsia species were known in South America: (i) Rickettsia rickettsii, transmitted by the ticks Amblyomma cajennense, and Amblyomma aureolatum, reported in Colombia, Argentina, and Brazil, where it is the etiological agent of Rocky Mountain spotted fever; (ii) Rickettsia prowazekii, transmitted by body lice and causing epidemic typhus in highland areas, mainly in Peru; (iii) Rickettsia typhi, transmitted by fleas and causing endemic typhus in many countries. During this new century, at least seven other rickettsiae were reported in South America: Rickettsia felis infecting fleas and the tick-associated agents Rickettsia parkeri, Rickettsia massiliae, Candidatus"Rickettsia amblyommii,"Rickettsia bellii, Rickettsia rhipicephali, and Candidatus"Rickettsia andeanae." Among these other rickettsiae, only R. felis, R. parkeri, and R. massiliae are currently recognized as human pathogens. R. rickettsii is a rare agent in nature, infecting < or =1% individuals in a few tick populations. Contrastingly, R. parkeri, Candidatus"R. amblyommii," R. rhipicephali, and R. bellii are usually found infecting 10 to 100% individuals in different tick populations. Despite rickettsiae being transmitted transovarially through tick generations, low infection rates for R. rickettsii are possibly related to pathogenic effect of R. rickettsii for ticks, as shown for A. aureolatum under laboratory conditions. This scenario implies that R. rickettsii needs amplifier vertebrate hosts for its perpetuation in nature, in order to create new lines of infected ticks (horizontal transmission). In Brazil, capybaras and opossums are the most probable amplifier hosts for R. rickettsii, among A. cajennense ticks, and small rodents for A. aureolatum.
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              Differentiation of spotted fever group rickettsiae by sequencing and analysis of restriction fragment length polymorphism of PCR-amplified DNA of the gene encoding the protein rOmpA.

              Currently, the genotypic identification of the spotted fever group (SFG) rickettsiae is based on restriction fragment length polymorphism analysis of PCR-amplified genes coding for the enzyme citrate synthase and the surface proteins rOmpA and rOmpB. A set of useful restriction endonucleases was found following comparison of Rickettsia rickettsii and R. prowazekii sequences. However, by using three PCR amplifications and four enzyme digestions with this set, it was impossible to differentiate between all of the known serotypes of the SFG rickettsiae. We amplified by PCR and sequenced using an automated laser fluorescent DNA sequencer a fragment of the gene encoding the protein rOmpA from 21 serotypes of the SFG rickettsiae. A 632-bp amplification product was obtained for most of the strains, although no product could be obtained by using R. akari, R. australis, R. helvetica, and R. bellii DNAs. We found a characteristic sequence for all strains studied except the two isolates of R. massiliae, isolates GS and Mtul. Using the software package BISANCE, we determined the restriction map of this fragment and identified five potentially useful endonucleases, RsaI, AluI, PstI, XbaI, and AvaII. We confirmed the computer analysis-derived profiles by PCR-restriction fragment length polymorphism analysis. The combination of the profiles obtained after digestion of the PCR product by RsaI and PstI allowed for the differentiation of 16 strains. The use of AluI and XbaI allowed for the characterization of R. parkeri and strain HA-91, respectively. R. africae and strain S were differentiated by AvaII digestion. Thus, using a single PCR amplification, we were able to differentiate all of the SFG rickettsiae whose ompA gene was amplified by PCR.
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                Author and article information

                Journal
                Parasit Vectors
                Parasit Vectors
                Parasites & Vectors
                BioMed Central
                1756-3305
                2014
                5 January 2014
                : 7
                : 7
                Affiliations
                [1 ]Department of Preventive Veterinary medicine and animal Health, Faculty of Veterinary Medicine, University of São Paulo, São Paulo, SP 05508-270, Brazil
                [2 ]Veterinary and Technical Responsible by the Association Fazenda Vila Real de Itu - Rodovia Marechal Rondon Km 113.5, Itu, SP 13312-901, Brazil
                Article
                1756-3305-7-7
                10.1186/1756-3305-7-7
                3892071
                24387674
                99779192-0bd5-4717-a5ba-e568c36d4ea7
                Copyright © 2014 Krawczak et al.; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 23 October 2013
                : 1 January 2014
                Categories
                Research

                Parasitology
                rickettsia rickettsii,brazilian spotted fever,amblyomma cajennense,capybara
                Parasitology
                rickettsia rickettsii, brazilian spotted fever, amblyomma cajennense, capybara

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